This project aims to understand high-speed chromosomal DNA replication, the central mechanisms of which are conserved across evolution. The polymerases that replicate chromosomes consist of three components: polymerase/exonuclease enzymes, a sliding clamp protein that holds the polymerases onto DNA and enables highly processive replication of both strands, and an ATP-dependent clamp loader complex that loads the clamp onto DNA. This project first established the principle that the bacterial sliding clamp (beta clamp) is a ring-shaped protein that encircles DNA. Completed work on this project has also shown that the sliding clamps from bacteria, eukaryotes (PCNA) and bacteriophage T4 (gene 45 protein) all share a common architecture. In addition, the structure of an active clamp loader assembly from E. coli (gamma complex) has been determined by X-ray crystallography in the absence of nucleotides, as has the structure of one subunit of the clamp loader bound to an open form of the beta clamp. The present proposal aims to extend our understanding of clamp loader mechanism by determining, by X-ray crystallography, the nature of the ATP-dependent conformational changes in the clamp loader, as well as the structure of the clamp loader in complex with the clamp and with DNA. Structural studies will also be extended to the clamp loader systems from other bacteria, to better understand conformational variability in the system. The segmental flexibility of this ATP-dependent machine will be studied by molecular dynamics simulations. The dynamics of the system will also be studied experimentally, using fluorescence resonance energy transfer and time resolved fluorescence polarization anisotropy decay from fluorophors linked to engineered cysteine residues. An active eukaryotic clamp loader (RFC) complex from the yeast Saccharomyces cerevisiae has been reconstituted from bacterially expressed protein, and purified in high yields. This RFC complex, similar in sequence to the human clamp loader, will be crystallized, both in isolation and in complex with the PCNA sliding clamp and DNA. The structural information resulting from this work will set the stage for the development of specific inhibitors of replication, either for the replicases from pathogenic bacteria, or for the human proteins with implications for cancer.